The broad objective of this research proposal is to throw light on the molecular mechanism of Na and K transport by biochemical and biophysical studies of two systems: (1) purified Na,K-ATPase preparations, one from the electroplax of electrophorus electricus, and the other from the rectal gland of Squalus acanthias; and (2) reconstituted Na and K transport in Na,K-ATPase liposomes. Part (1) will include: (a) determination of the stoichiometric ratio of the catalytic subunit to the glycoprotein subunit by alkylation of the cysteine residues with radioactive iodoacetate; (b) sequencing of the amino acids at the active site by tagging the active site by conversion of the aspartyl-beta-phosphate to (3H)homoserine; (c) the use of aryl azide derivatives of fatty acid and of ethanolamine ("photorulers") for determination of the penetration of the catalytic subunit and glycoprotein into the bilayer by photolabelling: and (d) optical diffraction of electron micrographs of negatively stained preparations of isolated catalytic subunit, which shows "crystallinity". Part (2) will include: (a) the effects of lectins on Na,K-ATPase activity and reconstituted Na and K transport in Na,K-ATPase liposomes in the hopes of throwing light on the role of the glycoprotein in catalytic activity and transport; (b) determination of the potential differences across the Na,K-ATPase liposomes in the hopes of throwing light on the role of the glycoprotein in catalytic activity and transport; (b) determination of the potential difference across the Na,K-ATPase liposomes during Na and K transport and the factors responsible for a pontential difference (electrogenic? diffusion potential?); (c) influence of the head groups and fatty acid chains in phospholipids on catalytic activity and reconstituted transport; (d) effects of limited proteolysis on the other surface of Na,K-ATpase liposomes; (e) a search for ionophoric activity in the Na,K-ATPase holoenzyme, in the "native" glycoprotein subunit, in proteolipids from the enzymes, and in fragments of the Na,K-ATPase prepared by various means; and (f) characterization and kinetic studies of an "AMP analogue" which may be an allosteric regulator of the Na,K-ATPase.